Methods: Here, we present the clinical and molecular characterization of a Tunisian patient with NS. A comprehensive mutations analysis of 29 genes belonging to the RAS pathway or encoding for interactors was performed, using targeted next generation sequencing.

Results: The results revealed a novel pathogenic substitution affecting the LZTR1, whose mutations have been described only in 5 cases of NS.

Conclusion: This report supports the implication of LZTR1 in Noonan syndrome. Next Generation Sequencing seems a suitable method for mutation detection in clinically and genetically heterogeneous syndromes such as NS.

Here we report a new case of a Tunisian patient with Noonan syndrome caused by LZTR1 mutation.

Case Presentation

The patient, a 6-year-old Tunisian boy, was the second child of healthy unrelated parents aged 26 (mother) and 41 years (father). The family history was unremarkable. His two brothers were healthy. Pregnancy and delivery were normal, and the boy was born at term. His birth weight was 3300 g, his length 49 cm and his head circumference 33 cm. He sat alone at 2½ years, walked at 4 years 10 months and had speech delay.

The Ion Torrent PGM system was used to sequence exons and splicing sites of 29 genes of the RAS MAPK signaling pathway involved in Noonan syndrome and other rasopathies, i.e.,: the commonly mutated PTPN11, SOS1, RAF1, KRAS, BRAF, NRAS, HRAS, MAP2K1, MAP2K2, SHOC2, CBL, SPRED1 genes and less common ones such as LZTR1 (more details about the complete list of genes are provided under request). The library kit was made with Agilent Haloplex technologies. The sequence analysis software was VarAFT (http://varaft.eu). Prediction of functional effects of nsSNPs was done with UMD predictor, MutationTaster and PolyPhen. Variants of interest were verified using the Integrative Genomics Viewer and validated by bidirectional Sanger Sequencing.

Results

A total of 163 variants were detected across the 29 genes analyzed. Filtering these results using in silico software predictors of mutation’s impact revealed only one heterozygous variant as potentially pathogenic: a heterozygous missense mutation in exon 4 of the LZTR1 gene predicted to lead to a missense amino acid change (NM_006767:c.347C>T, p.Ala116Val). This alteration was validated using Sanger sequencing in proband’s and available relatives, showing that it appeared de novo(Figure 2).

Discussion

In this study, we report a case of NS with typical clinical findings, harboring a mutation in LZTR1 (Leucine-Zipper-like Transcriptional Regulator-1), a gene rarely associated with NS.

LZTR1, located at 22q11.21, encodes a protein member of the BTBKelchsuperfamily implicated in several fundamental cell processes. The implication of LZTR1 in human disease was first reported with the DiGeorge Syndrome, as it was deleted in the majority of DiGeorge Syndrome patients [13]. More recently, somatic mutations with loss of heterozygosity in LZTR1 and germline loss-of-function variants in LZTR1 were respectively associated with glioblastoma multiforme [14] and schwannomatosis [15,16].

Germline LZTR1 variants in Noonan syndrome patients were first reported by Chen et al. in [17]. The authors performed Next Generation Sequencing in a cohort of 27 NS patients without a known NS gene mutation. Two of these patients had LZTR1 variants (p.R237Q and p.A249P) which were not considered as responsible for the NS phenotype, since the authors considered LZTR1 as a gene already associated with DiGeorge syndrome. In 2015, Yamamoto et al. identified rare variants of LZTR1 using whole-exome sequencing in 6/50 Brazilian probands (p.G248R, p. R284C, p.H287Y, p.Y119C, p.I647Vand p.F447L) and one Polish family ( p.S247N ) with NS and lacking mutation in the known NS genes [18]. Two of these variants were considered nonpathogenic because of their presence in unaffected relatives (p.F447L) or the weak of in silico pathogenicity prediction (p.I647V). The remaining five variants, p.G248R, p. R284C, p.H287Y, p.Y119C and p.S247N, were predicted to cause NS as they segregated with the NS phenotype or were de novo events and predicted to be deleterious by in silico analysis. Moreover, the LZTR1 variants identified in Brazilian patients were found in only 1/107 control cohort supporting their implication in NS. All the reported LZTR1 variants are localized in highly conserved kelch (KT) domain and are predicted to disrupt protein function. Kelch domain shape may also be responsible for binding to other proteins [19,20]. The missense heterozygous variant found in LZTR1 in our patient is localized in KT1 domain. In silico analysis predicted pathogenicity. Analysis of a control database of WES in 50 Tunisian patients affected by other disorders showed no LZTR1 variant. The mechanism by which mutations in LZTR1 causes NS is still unknown. Yamamoto et al. [18] suggested that missense heterozygous variants in LZTR1 may cause dysregulation of the RAS/MAPK pathway by increasing ERK signaling through a loss of tumor suppressor function.

The clinical findings of NS patients with LZTR1 variants were similar to PTPN11 positive individuals with the exception of short stature which was not frequent in the Brazilian cohort [19]. Our patient, contrary to what has been reported, had a short stature at -4,3 SD (Standard Deviation).

Table 1, updated from Yamamoto et al. [18], summarizes the clinical features seen in reported cases of NS patients with LZTR1 variants and highlights similarities between clinical findings seen in the 5 previously reported cases and in our case.

Our case further supports the implication of LZTR1 in the pathogenesis of NS. Nevertheless, functional studies are required to unravel the precise implication of LZTR1 variant of p.Ala116Val in Noonan syndrome.

Conclusion

The identification of causative mutations that underlie genetically heterogeneous syndromes such as Noonan Syndrome has been greatly facilitated by the emergence of high throughput sequencing. In this context, Targeted NGS methods can be used as a cost effective first line genetic test for confirmation of NS cases. Thus, an early and accurate genetic diagnosis and suitable management of patients will be possible. The case we report supports the involvement of LZTR1 in the pathogenesis of typical Noonan syndrome.

Acknowledgments

We thank the patient and his family for participating in this research. We would like to thank also both the personnel of the Department of Intensive Care and Neonatal Medicine, CHU Fattouma Bourguiba, Monastir, Tunisia and all members of the team of the INSERM unit UMR_S910, GMGF, Aix Marseille University, Marseille, France (Karim Harhouri, Cathy Bartoli, Guy Longepied, Françoise Merono…). This study was partially funded by the University Foundation A*MIDEX.

Competing Interests

The authors declare that they have no competing interests.

Author Details

Unit research 01/UR/08-14 Faculty of Medecine of Monastir, University of Monastir, Monastir 5000, Tunisia.